Dysregulated ceramide metabolism in mouse progressive dermatitis resulting from constitutive activation of Jak1

Coordinated lipid metabolism contributes to maintaining skin homeostasis by regulating skin barrier formation, immune reactions, thermogenesis, and perception. Several reports have documented the changes in lipid composition in dermatitis, including in atopic dermatitis (AD); however, the specific mechanism by which these lipid profiles are altered during AD pathogenesis remains unknown. Here, we performed untargeted and targeted lipidomic analyses of an AD-like dermatitis model resulting from constitutive activation of Janus kinase 1 (Spade mice) to capture the comprehensive lipidome profile during dermatitis onset and progression. We successfully annotated over 700 skin lipids, including glycerophospholipids, ceramides, neutral lipids, and fatty acids, many of which were found to be present at significantly changed levels after dermatitis onset, as determined by the pruritus and erythema. Among them, we found the levels of ceramides composed of nonhydroxy fatty acid and dihydrosphingosine containing very long-chain (C22 or more) fatty acids were significantly downregulated before AD onset. Furthermore, in vitro enzyme assays using the skin of Spade mice demonstrated the enhancement of ceramide desaturation. Finally, we revealed topical application of ceramides composed of nonhydroxy fatty acid and dihydrosphingosine before AD onset effectively ameliorated the progression of AD symptoms in Spade mice. Our results suggest that the disruption in epidermal ceramide composition is caused by boosting ceramide desaturation in the initiation phase of AD, which regulates AD pathogenesis.

The skin is composed of the epidermis, dermis, and subcutaneous tissue. The epidermis provides a primary barrier to external stimuli and antigens. Defects in epidermal barrier function are risk factors for various cutaneous disorders, such as infectious diseases, ichthyosis, and atopic dermatitis (AD) (1)(2)(3). In the epidermis, keratinocytes constitute four layers (stratum basale, stratum spinosum, stratum granulosum, and stratum corneum) and play pivotal roles in maintaining the epidermal barrier. Keratinocytes proliferate in the stratum basale and migrate outwards by differentiating into layer-specific cell types. During differentiation, keratinocytes develop a barrier function by forming tight junctions and producing lamellar lipids. In the stratum corneum, lipid lamellae surround corneocytes, the terminally differentiated keratinocytes, to form a hydrophobic barrier. Thus, the proliferation and differentiation of keratinocytes should be appropriately regulated to maintain normal epidermal barrier function and skin homeostasis.
Various lipid molecules contribute to the regulation of keratinocytes and the maintenance of epidermal barrier function. Lipid lamellae in the stratum corneum function as hydrophobic barriers. The major components of lipid lamellae are cholesterol, FAs, and ceramides, among which ceramides are the most abundant (4). Ceramides are composed of FA and longchain base (LCB) with structural variations in the number of double bonds, hydroxy groups, and carbon chain length and written by a combination of abbreviations corresponding to precursor structure (5-7) (supplemental Fig. S1). For example, the major ceramide classes in murine skin are composed of nonhydroxy FA and sphingosine or dihydrosphingosine written as Cer [NS] or Cer [NDS]. Lipid lamellae have a unique ceramide composition with a high proportion of very long-chain FA (VLCFA)-containing ceramides and acylceramides with an additional hydrophobic chain (linoleic acid) (8). Dysfunction of the enzymes responsible for producing lamellar ceramides causes severe barrier defects in humans and mice (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). For example, mutations in the gene encoding patatin-like phospholipase domain-containing 1 (PNPLA1), which plays a pivotal role in the biosynthesis of acylceramide, cause autosomal recessive congenital ichthyosis (21). Pnpla1 knockout mice show neonatal lethality due to epidermal permeability barrier defects (17,21,22). Although ceramides have received significant attention for a long time, other lipid species, such as triacylglycerol, ethanolamine plasmalogen, and LCB, also play important roles in maintaining barrier function and skin homeostasis (22)(23)(24)(25)(26). Thus, the skin lipid metabolic network elaborately regulates epidermal homeostasis as each lipid molecule exerts a characteristic bioactivity or physiological function.
AD is a chronic inflammatory dermatitis characterized by barrier disruption, pruritus, and excessive activation of type 2 immune response. Abnormalities in tight junctions and dysregulated keratinocyte differentiation are observed in AD (27,28). In addition, diverse lipid metabolic changes are observed in AD lesional skin. For example, changes in the carbon chain length of lipids, such as ceramides, sphingomyelins (SMs), lysophosphatidylcholines (LPCs), or FAs, are observed in AD lesional skin, and the composition of ceramide classes is altered (29)(30)(31)(32). However, the specific mechanism by which the lipid profiles are altered during the pathogenesis of AD remains unknown.
In this study, we performed untargeted and targeted lipidomic analyses to determine the global lipid profiling in a progressive AD-like dermatitis of Spade mice that can spontaneously develop pruritic dermatitis by the mutation of the gene encoding Janus kinase 1 (Jak1), causing constitutive activation of Jak1 signaling (33). Notably, VLCFA-containing Cer[NDS] were selectively reduced with enhanced ceramide desaturation in the skin before the onset of AD symptoms, and topical application of Cer[NDS] effectively ameliorated the progression of AD symptoms in Spade mice.

Experimental animals
Spade mice were kindly provided by H. Koseki (RIKEN Center for Integrative Medical Sciences, Laboratory for Developmental Genetics). Systemic Jak1 mutation in Spade mice was produced by N-ethyl N-nitrosourea (ENU) mutagenesis protocol. The detailed method was described previously (33). Mice were maintained in specific pathogen-free environment with a 12-h light/dark cycle with water and a standard diet (CLEA Rodent Diet CE-2; CLEA Japan, Tokyo, Japan) provided ad libitum. All experiments involving the use of animals were approved by and performed in accordance with the Guidelines for Animal Experimentation of the Animal Use Committee at Keio University.

Skin permeability assay
The skin permeability barrier was evaluated by measuring transepidermal water loss (TEWL) in the ears of mice using an evaporimeter (AS-VT100RS; Asahi Biomed, Yokohama, Japan). Measurements were taken three times for each ear, and the average value was calculated.

Preparation of epidermis of murine ear
Murine ears were separated using forceps and incubated in RPMI containing 10% FBS, phosphatase inhibitor (Phosstop; Merck Millipore, Burlington, MA, USA), cOmplete protease inhibitor (Merck Millipore), and 1 mg/ml dispase (Thermo Fisher Scientific, Waltham, MA, USA) for 80 min at 37 • C. After incubation, the epidermis was carefully separated from the dermis.

LC-MS/MS analysis
An ACQUITY UPLC system (Waters, Milford, MA, USA) coupled with TripleTOF 6600 (Sciex, Framingham, MA, USA) was used for performing untargeted lipidomics. The conditions for LC separation and electrospray ionization were previously described (34). MS-DIAL was used for peak picking and alignment.

Quantitative RT-PCR
The frozen tissue was pulverized with a metal cone using a MULTI-BEADS SHOCKER MB1200. RNeasy Mini Kit (Qiagen, Venlo, Netherlands) was used for total RNA preparation. 500 ng of RNA was subjected to reverse transcription using ReverTra Ace qPCR RT Master Mix (TOYOBO, Osaka, Japan). THUNDERBIRD NEXT SYBR qPCR MIX (TOYOBO) was used for quantitative RT-PCR. The forward (F) and reverse (R) primer pairs used in this study are listed in supplemental Table S3.
The transmission electron microscopic analysis of lipid lamellae using ruthenium tetroxide was conducted as described previously (21,36). For transmission electron microscopy, mouse ear skin was fixed with 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M cacodylate buffer at 4 • C. Then, samples were fixed in 2% osmium tetroxide solution at 4 • C for 2 h and subsequently postfixed in 2% ruthenium tetroxide solution and 0.25% potassium hexacyanoferrate at 4 • C for 2 h under pressure. After dehydration in graded ethanol solutions (30-100%), samples were infiltrated with propylene oxide three times for 15 min. Then, samples were embedded in the epoxy resin (EPON812; EM Japan, Tokyo, Japan). After making ultrathin ear sections (80-90 nm) by 2088 ULTRATOME Ⅴ (LKB, Vienna, Austria), samples were stained with 2% uranyl acetate for 15 min and lead stain solution (Merck Millipore) for 2 min. Transmission electron microscopy observation was conducted by H-7600 (HITACHI, Tokyo, Japan). Images were taken at 100 kV of acceleration voltage.

Analytical validation
Results are expressed as the mean + standard error. Data were analyzed using Excel and R statistical software. They were statistically analyzed using Student's t-test, Dunnett's test, and Mann-Whitney's U test. P < 0.05 was considered to be statistically significant.

Global lipid profiling during AD pathogenesis in Spade mice
Hyperactivation of Jak1 caused by the single amino acid substitution in Spade mice leads to Th2 dermatitis (33). Progressive dermatitis develops as desquamation and redness of the ears at approximately 8 weeks of age. At 10 weeks of age, serum IgE and IgG1 levels are increased, and Th2 cytokines, such as IL-4, IL-5, and IL-13, produced by CD4 + cells are upregulated, followed by elevated serum histamine levels at 12 weeks of age. Skin lesions manifested as epidermal hyperplasia at 8 weeks, while there were few morphological changes at 4 weeks of age (Fig. 1A). Transmission electron microscopic analysis showed that in Spade mice at 4 weeks of age, normal lipid lamellar structures were observed in most parts of the sections, as shown in WT. However, some aggregates, like remaining substances of lamellar bodies, were found in the Spade lipid lamellae occasionally (Fig. 1B). TEWL, the barrier function readout, was significantly elevated in Spade mice at 4 weeks of age (Fig. 1C), suggesting that barrier defects had occurred before disease onset. To analyze the comprehensive lipid profile at each stage of pathogenesis, WT and Spade skin tissues at P0, 4, 8, and 10 weeks of age were applied to untargeted lipidomics, which resulted in monitoring 745 lipid species annotated by negative and positive ion modes (supplemental  (32). At 10 weeks of age, the mRNA expression of Elovl6 was reduced significantly and Elovl3/4/5 levels tended to decrease compared to WT (supplemental Fig. S2H). Interestingly, in Spade mice at 4 weeks of age, we found a cluster with a large reduction in ceramide species, although there was no obvious change in other lipid classes (Fig. 1D). Ceramides are classified into various classes according to the different combinations of their precursors (LCB and FA) (5-7) (supplemental Fig. S1). The volcano plot showed a dramatic and significant decrease in the Cer[NDS] species in Spade mice at 4 weeks of age, before the onset of AD (Fig. 1E) Table S4).

VLCFA-containing Cer[NDS] were selectively reduced in the Spade epidermis before AD onset
Ceramides have a crucial role in regulating skin barrier function, and it has been shown that the disruption of ceramide metabolism leads to skin inflammation (37,38). Since ceramide metabolism was altered in Spade mice before AD onset, we focused on its metabolism. To ascertain whether the disruption of ceramide metabolism occurred in the epidermal or dermal tissue of the ear, we performed MRM-based targeted analysis for the quantification of Cer[NS] and Cer[NDS] containing C16-18 LCBs and C14-32 FAs in the epidermis and dermis prepared from WT and Spade mice at 4 weeks of age. In Cer[NS] with d16:1 or d18:1 LCBs, ceramides with C24 or more FAs dominantly exited the epidermis, whereas those with C18 or fewer FAs were dominant in the dermis (Fig. 2A).   (Fig. 2D). In addition, the amount of dihydrosphingosine (DHS), a precursor of Cer[NDS], declined significantly (d16:0, 13%; d17:0, 8.8%; d18:0, 17% vs. WT) in the Spade epidermis compared with WT, and the amount of sphingosine (Sph) also declined (d16:1, 62%; d17:1, 46%; d18:1, 64% vs. WT) (supplemental Fig. S3A). The levels of DHS (d18:0) and Sph (d16:1, d17:1, and d18:1) in the dermis did not change (supplemental Fig. S3B). In the Spade epidermis, the total amount of Cer[NDS] declined significantly, although that of Cer[NS] did not change (supplemental Fig. S3C,  D). Furthermore, the total amount of SM containing Sph d18:1, the end product of Cer[NS], was significantly upregulated (139% vs. WT) (supplemental Fig. S3E). Altogether, VLCFA-containing Cer[NDS] were selectively reduced in the epidermis, suggesting that aberrant sphingolipid metabolism might impair skin homeostasis in Spade mice before the onset of AD.

Degs1-associated aberrant ceramide metabolism in Spade mice
We next performed in vitro measurements of the enzyme activities to investigate the mechanism by which Cer[NDS] were selectively decreased in Spade mice. In the sphingolipid metabolic pathway, acyl-CoA is condensed with serine to generate 3-ketoDHS by serine palmitoyl transferase (SPT), which is reduced to DHS by 3-ketoDHS reductase (KDSR) (39) (Fig. 3A).

DHS is acylated to Cer[NDS] via ceramide synthase (CERS). Subsequently, Cer[NDS] are reduced to Cer[NS]
by DEGS. We investigated whether the reduction of Cer [NDS] in Spade mice is attributed to either decreased de novo synthesis (Spt, Kdsr), decreased ceramide synthesis (CerS), or increased ceramide desaturation (Degs). We   measured the de novo synthesis activity using C16:0-CoA and 13 C 3 , 15 N-serine as substrates. As a result, the production of DHS ( 13 C 2 , 15 N-d18:0) was not changed in the Spade mice tissue at 4 weeks of age compared to WT (Fig. 3B). This result showed that de novo synthesis activity did not decrease in 4-week-old Spade mice compared to WT. CerS activity was measured using C24:0-CoA, which is recognized by CerS2/3 as a substrate (40,41). When d 7 -Sph (d 7 -d18:1) was incubated with C24:0-CoA, there was no difference in the production of Cer[NS] d 7 -d18:1/24:0 between the WT and Spade mice tissue homogenates (Fig. 3C). Similarly, the production of Cer[NDS] d 7 -d18:0/24:0 did not change when d 7 -DHS (d 7 -d18:0) was used, suggesting that the CerS activity was not reduced in Spade mice (Fig. 3D) (Fig. 3D, E). We also used C16:0-CoA, which is recognized by CerS5/6 for the measurement of CerS and desaturation, and found normal CerS and enhanced ceramide desaturation (42, 43) (supplemental Fig. S4A-C). These results indicate the upregulation of ceramide desaturation and could explain why the amount of Cer [NDS] was reduced in the Spade skin before AD onset. To unveil the mechanism of ceramide desaturation enhancement in Spade mice, we investigated the expression of epidermal genes related to ceramide metabolism. The mRNA levels of Degs1, involved in ceramide desaturation, were not significantly upregulated, although some sphingolipid metabolic genes were slightly downregulated (Sptlc3, 61%; Sgpp1, 71%) in the Spade epidermis at 4 weeks of age (Fig. 3F). In addition, there was no significant change in Degs1 protein expression in Spade mice at 4 weeks of age, whereas ceramide desaturation was enhanced approximately 2-fold in Spade mice compared with WT (Figs. 2D and 3G).

DISCUSSION
In this study, using global lipidome profiling, we unveiled the dysregulated sphingolipid metabolism observed during dermatitis progression caused by the constitutive activation of Jak1. Cer[NDS] with VLCFAs (C22 or more) were drastically downregulated in Spade mice before disease onset. These alterations illustrate the potentially novel pathogenic function of Jak1dependent dermatitis pathology.
We used a mouse model of Jak1 activity-dependent dermatitis to understand the pathogenesis of dermatitis at the molecular level. JAK inhibitors have already been approved as human AD drugs, and many JAK inhibitors have advanced to clinical studies (44). Two reports have indicated that increased Jak1 activity led to an inflammatory skin phenotype in mice (45,46). Therefore, it is suggested that Jak1 is important for maintaining skin homeostasis. In Spade mice, the homozygous R878H mutation in the kinase domain leads to the activation of Jak1 (33). This mouse model showed enhanced phosphorylation of Stat proteins in the upper epidermis. Furthermore, bone marrow chimera experiments revealed that the development of dermatitis required the expression of the Spade allele by nonhematopoietic cells. Altogether, these results suggest that excessive Jak1 activation in the epidermis is involved in the pathogenesis of AD. Further, we clarified that in the epidermis, Cer[NDS] containing VLCFAs were downregulated in the condition of Spade mice inflammation mediated by Jak1 activation. There is no previous report describing the relationship between the JAK1 pathway and ceramide metabolism. Although some reports show that the cytokine, activating broad pathways/signals including the JAK1 pathway, altered ceramide metabolism in vitro experiments in keratinocytes, JAK1-dependency is not addressed (32,(47)(48)(49). Thus, this is the first report showing dysregulated ceramide metabolism via constitutive activation of Jak1 in progressive dermatitis in mice.
The skin lipid metabolic network elaborately regulates epidermal homeostasis by each lipid molecule exerting its characteristic bioactivity or physiological function. Dysregulated lipid metabolism is observed in the skin of patients with AD. However, it remains unknown how lipid profiles are altered during the pathogenesis of AD. In this study, we demonstrated the global lipid profiling accompanying dermatitis development using untargeted and targeted lipidomic analyses. After dermatitis onset in Spade mice, various lipid metabolic changes occurred, similar to lipid changes in the human AD region, especially in ceramide, LPC, and FA (supplemental Fig. S2C, D, F, and G). In addition, these lipid species showed a proportional change in carbon chain length, that is, a decline in VLC saturated lipids ( In fact, VLCFA and ceramide metabolic aberrancy cause skin barrier disruption, resulting in neonatal lethality in mice and parakeratosis in humans (9,12,15,16,(49)(50)(51). Thus, in Spade mice after AD onset, aberrant VLC-lipid composition would rupture the lipid lamellar order, triggering further exacerbation of the barrier function and disease. Berdyshev et al. showed the gene expression pattern of ELOVL family members 1-7 in the epidermis of patients with AD, indicating that the mRNA levels of ELOVL3/6 are downregulated in the human AD region. (32). ELOVL3 forms LC and VLC FAs (C18-C24) from their short-chain precursors, whereas ELOVL6 forms LC FAs (C12-C18) (52,53). They showed that ELOVL3/6 contributed to the decline in VLC lipids (C24:0 in all glycerolipids, Cer[NS] 42:1;O2-44:1;O2, and SM 42:1;O2) and an increase in LC lipids (C16:0 in all glycerolipids) in keratinocytes. In the ear of Spade mice at 10 weeks of age, the level of Elovl6 was markedly reduced, and Elovl3/4/5 showed a decreasing trend (supplemental Fig. S2H). ELOVL4, which is essential for skin barrier function, is responsible for the formation of VLCFAs (C28 or more), whereas ELOVL5 is responsible for the formation of LC polyunsaturated FAs (52,53). Therefore, it is possible that in Spade mice after AD onset, decreased levels of Elovl3/4/6 are responsible for the change in the chain length of lipids (Cer[NS], LPC, and FA).
Before dermatitis onset in Spade mice at 4 weeks of age, VLCFA-containing Cer[NDS] were selectively reduced in the epidermis ( Fig. 2A-C). Considering VLCtype reduction, we speculated that the enzyme activity of Elovl4 and/or CerS3 was affected. However, we assume the activity of Elovl4 is not downregulated in Spade mice at 4 weeks of age because the chain length of fatty acyls in Cer[NS] and free FAs are not changed. Spt could be also affected because DHS content was decreased in the Spade epidermis (supplemental Fig. S3A). Further, Cer [NDS] reduction could be attributed to the increased activity of Degs1, an enzyme responsible for Cer [NDS] desaturation. Thus, we measured these enzyme activities using skin homogenates and found that only Degs1 activity was upregulated (Fig. 3B-D). Δ4-dihydroceramide desaturase DEGS1 is the enzyme that converts Cer  (Fig. 2C, D). The reduced amount of Cer[NDS] (30 nmol/ mg tissue vs. WT) in Spade mice appears to be reflected in the increased amount of SM (90 nmol/mg tissue vs. WT), the endo product of Cer[NS] (supplemental Fig. S3C-E) (57). These VLC ceramides, which form lipid lamellae, are synthesized in differentiated keratinocytes in stratum spinosum or stratum granulosum. When DEGS1 activity was enhanced in the upper epidermis, VLCFA-containing Cer[NDS] levels would be selectively reduced.
Wigger et al. established an in vitro protein assay using rat liver microsomes to monitor de novo synthesis of sphingolipid by using deuterium-labeled chemicals (58). Using this method, we analyzed the activities of sphingolipid metabolic enzymes in tissues. We found that ceramide desaturation was enhanced approximately 2-fold higher in Spade tissue homogenates than in WT with no change in Degs1 protein expression level (Fig. 3E, G), suggesting that the enhancement of ceramide desaturation activity was attributed to its relative activity rather than protein expression level. DEGS1 is an ER membrane protein that contains three consensus motifs characteristic of membrane lipid desaturases (59)(60)(61). DEGS1 enhances its enzyme activity by myristoylation of the N-terminal domain (62). In addition, DEGS1 is phosphorylated at the 307 serine residue, although the effect of phosphorylation on enzyme activity remains unknown (63). Any posttranslational modifications may enhance the activity of Degs1 in Spade mice.
In Spade mice at 4 weeks of age, TEWL increased significantly (Fig. 1C). Further, although the normal lamellar structure was observed in Spade mice as well as WT, some aggregates, like the remaining substances of lamellar bodies, were found only in Spade lipid lamellae occasionally (Fig. 1B). These structures are observed in other skin barrier defect mice with lamellar lipid abnormalities such as Pnpla1 or Abhd5 knockout mice (21,36). Therefore, it is suggested that JAK1 activation and dysregulated ceramide metabolism led to the abnormal aggregates in the lipid lamellae, which contributed to the barrier abnormality exhibited in Spade mice at 4 weeks of age.
Topical application of Cer[NDS] significantly suppressed inflammatory gene expression and epidermal thickening without affecting skin barrier function evaluated by TEWL, resulting in ameliorating dermatitis onset and progression in Spade mice (Figs. 4A-G and S5A-E). Therefore, we speculated that topically applied Cer[NDS] affected local inflammatory status and keratinocyte proliferation/differentiation without affecting skin barrier function. We found that epidermal hyperplasia in Spade mice was suppressed by Cer[NDS] d18:0/24:0 application (Fig. 4F, G) Taken together, we revealed for the first time that dysregulated lipid metabolism, namely Degs1-associated aberrant ceramide metabolism, occurs during AD pathogenesis in Jak1-associated progressive dermatitis. Our results provide novel insights into the causal relationship between ceramide metabolism and homeostasis in the skin and propose that ceramide metabolic regulation is potentially useful for ameliorating cutaneous disorder.

Data availability
The data generated in this study are available from the corresponding author upon request. Dr Makoto Arita, Division of Physiological Chemistry and Metabolism, Graduate School of Pharmaceutical Sciences, Keio University, Tokyo, Japan. E-mail: arita-mk@pha. keio.ac.jp The raw mass spectrometric data has been deposited in "Metabolomics Workbench". To view dataset's webpage, go to www.metabolomicsworkbench.org. Study ID is "ST002195".

Supplemental data
This article contains supplemental data.